Abstract A14: A forward genetic screen for genes that cooperate with PTEN in lung cancer development

Abstract

Abstract Phosphatase and Tensin Homolog (Pten) negatively regulates the PI3K signaling pathway and functions as a tumor suppressor in many forms of cancer. The role of Pten in lung cancer is not well understood, although protein expression is frequently lost. Evidence from a mouse model has suggested that loss of Pten is not sufficient to induce lung tumor formation, but will cooperate with activated Kras to decrease latency and increase severity of lung tumors. In order to better understand the role of Pten in lung cancer we performed a forward genetic screen in mice aimed at identifying mutations that cooperate with Pten during lung cancer development. To target both loss of Pten and random mutations to lung tissue we generated mice containing a conditional Sleeping Beauty (SB) transposase allele (Rosa26-Stopfl/fl-SB11) and a conditional Pten allele (Ptenfl/fl). When crossed to mice expressing Cre recombinase under control of the lung-specific surfactant protein C (SPC) promoter, the SB11 gene is expressed while Pten is rendered inactive in lung tissue. In these Pten null cells, mutations are created by SB11 mediated transposition of a mutagenic transposon, T2/Onc, engineered to disrupt tumor suppressor genes or overexpress oncogenes. We have performed IHC for the SB transposase on lung tissue of mice harboring both Rosa26-Stopfl/fl-SB11 and SPC-Cre alleles and find the transposase is expressed in approximately 20% of lung epithelial cells. Experimental mice harboring all four transgenes (Ptenfl/fl, Rosa26-Stopfl/fl-SB11, SPC-Cre, and T2/Onc), along with two control groups (Ptenfl/fl, Rosa26-Stopfl/fl-SB11, and SPC-Cre or Ptenfl/fl, Rosa26-Stopfl/fl-SB11, and T2/Onc), were aged and monitored for morbidity. Median survival of the experimental and control group containing SPC-Cre (lung tissue is Pten null) was approximately 300 days, while median survival of the control group containing T2/Onc (lungs are wild-type for Pten) was 500 days (p < 0.0001). Upon necropsy, 29% of experimental animals, 12% of the Pten null control group, and 3% of the Pten wild-type control group harbored macroscopic lung tumors. Using linker-mediated PCR and high-throughput sequencing we sequenced and mapped over 10,000 transposon insertions in 31 lung tumors from 18 mice. Statistical analysis of these insertions identified 44 common insertion sites which indicate potential driver mutations for lung cancer. Candidate genes identified include known cancer genes (e.g., Map2k4 and Lpp), genes dysregulated in human lung tumors (e.g., Rlf and Car8) and genes not previously associated with lung cancer (e.g., Mllt10 and Ppp3ca). We will be testing the effect of knocking-down or over-expressing these candidate cancer genes in cell lines and mouse models to determine their role in tumor formation and development. The results of this study will be used to identify new diagnostic markers and therapeutic targets for treating lung cancer. Citation Information: Cancer Res 2009;69(23 Suppl):A14.